1. Field of the Invention
The present invention relates to antibody proteins that specifically bind fibroblast activation protein alpha (FAPxcex1). The invention also relates to the use of said antibodies for diagnostic and therapeutic purposes and methods of producing said antibodies.
2. Related Art
The invasive growth of epithelial cancers is associated with a number of characteristic cellular and molecular changes in the supporting stroma. A highly consistent molecular trait of the reactive stroma of many types of epithelial cancer is induction of the fibroblast activation protein alpha (from now on referred to as FAP), a cell surface molecule of reactive stromal fibroblasts originally identified with monoclonal antibody F 19 (Garin-Chesa, P., et al., xe2x80x9cCell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers,xe2x80x9d Proc. Natl. Acad. Sci. 87:7235 (1990)). Since the FAP antigen is selectively expressed in the stroma of a range of epithelial carcinomas, independent of location and histological type, a FAP-targeting concept has been developed for imaging, diagnosis and treatment of epithelial cancers and certain other conditions. For this purpose a monoclonal antibody termed F19 that specifically binds to FAP was developed and described in U.S. Pat. No. 5,059,523 and WO 93/05804, which are hereby incorporated by reference in their entirety.
One serious problem that arises when using non-human antibodies for applications in vivo in humans is that they quickly raise a human anti-non-human response that reduces the efficacy of the antibody in patients and impairs continued administration. Humanization of non-human antibodies is commonly achieved in one of two ways: (1) by constructing non-human/human chimeric antibodies, wherein the non-human variable regions are joined to human constant regions (Boulianne, G. L., et al., xe2x80x9cProduction of functional chimeric mouse/human antibody,xe2x80x9d Nature 312:643 (1984)) or (2) by grafting the complementarity determining regions (CDRs) from the non-human variable regions to human variable regions and then joining these xe2x80x9creshaped humanxe2x80x9d variable regions to human constant regions (Riechmann L., et al., xe2x80x9cReshaping human antibodies for therapy,xe2x80x9d Nature 332:323 (1988)). Chimeric antibodies, although significantly better than mouse antibodies, can still elicit an anti-mouse response in humans (LoBuglio, A. F., et al., xe2x80x9cMouse/human chimeric monoclonal antibody in man: Kinetics and immune response,xe2x80x9d Proc. Natl. Acad. Sci. 86:4220 (1989)). CDR-grafted or reshaped human antibodies contain little or no protein sequences that can be identified as being derived from mouse antibodies. Although an antibody humanized by CDR-grafting may still be able to elicit some immune reactions, such as an anti-allotype or an anti-idiotypic response, as seen even with natural human antibodies, the CDR-grafted antibody will be significantly less immunogenic than a mouse antibody thus enabling a more prolonged treatment of patients.
Another serious limitation relating to the commercial use of antibodies for diagnosis, imaging and therapy is their producibility in large amounts. In many instances recombinant expression of native, chimeric and/or CDR-grafted antibodies in cell culture systems is poor. Factors contributing to poor producibility may include the choice of leader sequences and the choice of host cells for production as well as improper folding and reduced secretion. Improper folding can lead to poor assembly of heavy and light chains or a transport incompetent conformation that forbids secretion of one or both chains. It is generally accepted that the L-chain confers the ability of secretion of the assembled protein. In some instances multiple or even single substitutions can result in the increased producibility of antibodies.
Because of the clinical importance of specific immunological targeting in vitro and in vivo of specific disease-related antigens for diagnosis and therapy in humans, there is a growing need for antibodies that combine the features of antigen specificity, low immunogenicity and high producibility.
Therefore, the problem underlying the present invention was to provide antibody proteins that combine the properties of specific binding to FAP, low immunogenicity in humans, and high producibility in recombinant systems.
The technical problem is solved by the embodiments characterized in the claims.
The present invention provides new antibody proteins having the complementary determining regions of the monoclonal antibody F19 (ATCC Accession No. HB 8269), said new antibody proteins specifically binding to fibroblast activation protein (FAP), characterized in that they have framework modifications resulting in the improved producibility in host cells as compared to a chimeric antibody having the variable regions of F19 and foreign constant regions.
As used herein, an xe2x80x9cantibody proteinxe2x80x9d is a protein with the antigen binding specificity of a monoclonal antibody.
xe2x80x9cComplementarity determining regions of a monoclonal antibodyxe2x80x9d are understood to be those amino acid sequences involved in specific antigen binding according to Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication No. 91-3242. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) in connection with Chothia and Lesk (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
As used herein, the term xe2x80x9cframework modificationsxe2x80x9d refers to the exchange, deletion or addition of single or multiple amino acids in the variable regions surrounding the individual complementarity determining regions. Framework modifications may have an impact on the immunogenicity, producibility or binding specificity of an antibody protein. xe2x80x9cFibroblast activation protein (FAP)xe2x80x9d, also designated fibroblast activation protein alpha (FAPxcex1), is a membrane-bound glycoprotein belonging to the serine protease gene family (WO 97/34927). No shed or secreted form of FAP is known. FAP can be characterized by its binding to the monoclonal antibody F19 (F19 is obtainable from the hybridoma cell line with the accession No. HB 8269 deposited at the ATCC).
The term xe2x80x9cfibroblast activation protein specific bindingxe2x80x9d of an antibody protein is defined herein by its ability to specifically recognize and bind FAP-expressing human cells. The binding specificity of the proteins of the invention can be determined by standard methods for the evaluation of binding specificity such as described in an exemplary fashion in examples 6, 8 and 12.
The term xe2x80x9cchimeric antibodyxe2x80x9d refers to an antibody protein having the light and heavy chain variable regions as described in FIGS. 17 and 18 and foreign constant regions. xe2x80x9cForeign constant regionsxe2x80x9d as defined herein are constant regions which are different from the constant regions of F19. For comparing an antibody protein of the invention to a chimeric antibody it is to be understood that such a chimeric antibody must contain the same constant regions as said antibody protein. For the purpose of demonstration and comparison alone the human constant heavy and light chains as described in FIGS. 19 to 22 are used in an exemplary fashion.
To provide the antibody proteins of the present invention, the nucleic acid sequences of the heavy and light chain genes of the murine antibody designated F19 were determined from RNA extracted from F19 hybridoma cells (ATCC Accession No. HB 8269).
In one embodiment the present invention relates to antibody proteins having the complementary determining regions of the monoclonal antibody F19 (ATCC Accession No. HB 8269), said new antibody proteins specifically binding to fibroblast activation protein (FAP), characterized in that they have framework modifications resulting in the improved producibility in host cells as compared to a chimeric antibody having the variable regions of F19 and foreign constant regions, wherein said antibody protein is derived from the murine antibody designated F19 (ATCC Accession No. HB 8269).
To generate humanized FAP-specific antibody proteins a chimeric antibody was constructed, having variable regions of the light and heavy chains of F19 and human light and heavy constant regions, respectively. The construction and production of chimeric mouse/human antibodies is well known (Boulianne et al. (1984), referenced above) and demonstrated in an exemplary fashion in examples 1 and 2.
The variable regions of the antibody proteins of the present invention are typically linked to at least a portion of the immunoglobulin constant region (FC), typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, but preferably immortalized B cells (see Kabat et al., supra, and WO 87/02671). Hence the antibody proteins of the invention may contain all or only a portion of the constant region as long as they exhibit specific binding to the FAP antigen. The choice of the type and extent of the constant region depends on whether effector functions like complement fixation or antibody dependent cellular toxicity are desired, and on the desired pharmacological properties of the antibody protein. The antibody protein of the invention will typically be a tetramer consisting of two light chain/heavy chain pairs, but may also be dimeric, i.e., consisting of a light chain/heavy chain pair, e.g., a Fab or Fv fragment.
Therefore, in a further embodiment the invention relates to antibody proteins according to the invention, characterized in that they have a variable light chain region and a variable heavy chain region, each joined to a human constant region.
In particular, the variable region of the light chain was joined to a human kappa constant region and the variable region of the heavy chain was joined to a human gamma-1 constant region. Other human constant regions for humanizing light and heavy chains are also available to the expert.
Therefore, in one particular embodiment the antibody proteins of the invention contain a human kappa constant region.
Also, in another particular embodiment the antibody proteins of the invention contain a human gamma-1 constant region.
One particular xe2x80x9cchimeric F19 antibodyxe2x80x9d protein (cF19) consists of the light and heavy chain variable and constant regions described in FIGS. 17 to 22. cF19 demonstrates specific binding and high avidity to the FAP antigen. As demonstrated in example 2, the expression of cF19 in COS cells (cells derived from the kidney of an African green monkey) is poor, ranging from about 10 to 60 ng/ml, which is at least 10 fold less than most antibodies.
In an attempt to increase expression levels of cF19, the leader sequence of the F19 VL region was changed by substitution of proline to leucine at position 9. This single change in amino acid in the leader sequence resulted in at least doubling the amount of chimeric antibody produced in COS cells. For the expression of this particular chimeric antibody in COS cells the following mutated leader sequence of the light chain: MDSQAQVLMLLLLWVSGTCG (SEQ ID NO:60, and the following leader sequence of the heavy chain: MGWSWVFLFLLSGTAGVLS (SEQ ID NO:61were used.
According to the invention the term xe2x80x9cimproved producibilityxe2x80x9d in host cells refers to the substantial improvement of expression levels and/or purified antibody yields when compared with the expression levels and/or antibody yields of a chimeric antibody without framework modifications as defined above. Two particular but not limiting examples for demonstrating improved producibility are exemplified for the COS cell expression system (in examples 2 and 5) and for the CHO cell expression system (in examples 10 and 11).
While the mutation of the leader sequence only leads to a doubling of the expression yield of the chimeric F19 antibody, a substantial improvement as defined herein refers to an improvement in expression level and/or purification yield of at least a factor of 10.
In a preferred embodiment, the invention refers to antibody proteins, characterized in that their expression levels in crude media samples as determined by ELISA and/or purified antibody yields exceed the expression levels and/or purification yields of the chimeric antibodies without framework modifications by at least a factor of 10.
In more preferred embodiment, the invention refers to antibody proteins, characterized in that their expression levels in crude media samples as determined by ELISA and/or purified antibody yields exceed the expression levels and/or purification yields of the chimeric antibodies without framework modifications by at least a factor of 20.
In a most preferred embodiment, antibody proteins, characterized in that their expression levels in crude media samples as determined by ELISA and/or purified antibody yields exceed the expression levels and/or purification yields of the chimeric antibodies without framework modifications by at least a factor of 100.
Improved producibility of the recombinant antibody proteins of the invention can be demonstrated for eukaryotic cells in general as shown for COS and CHO (Chinese hamster ovary derived cells) eukaryotic cells (see examples 5 and 11). In a further embodiment, the present invention relates to recombinant antibody proteins characterized in that they display improved producibility in eukaryotic cells.
In a preferred embodiment the present invention relates to antibody proteins, wherein said eukaryotic cell is a Chinese hamster ovary cell (CHO cell).
It was unexpectedly found that certain framework modifications of the light chain variable regions determine the improved producibility of the antibody proteins of the invention. Three versions of reshaped light chain variable regions, designated version A, B and C, as described in FIGS. 1 to 6, were prepared.
Light chain variable region versions A, B, and C demonstrate substantially improved producibility in CHO cells (see example 11). While light chain variable region versions A and C differ from light chain variable region version B by only two common amino acid residues they display an even further substantial improvement in producibility. There is at least another 10 fold difference in antibody secretion levels between the human reshaped F19 light chain version B and versions A or C. Reshaped human F19 light chain version A and B only differ in their amino acid sequences by two residues at positions 36 (Tyr to Phe mutation) and 87 (Tyr to Asp mutation) (nomenclature according to Kabat). This negative effect on the secretory capability of antibodies containing the light chain variable region version B could have been indirect if the Tyr to Asp and Tyr to Phe mutations, considered individually or together, merely caused improper folding of the protein. But this is unlikely to be the case since antigen binding assays show that immunoglobulins containing F19 light chain version B have similar avidities to those paired with F19 light chain version A or C, suggesting that they were not grossly misfolded.
Residue 87 in reshaped human F19 light chain version B seems particularly responsible for the reduction of secretion when compared to versions A and C.
In a preferred embodiment, the present invention relates to antibody proteins according to the invention, wherein the amino acid in Kabat position 87 of the light chain region is not asparagine.
In a more preferred embodiment, the invention relates to antibody proteins according to the invention, wherein the amino acid in Kabat position 87 of the light chain region is selected from aromatic or aliphatic amino acids.
In a most preferred embodiment, the present invention relates to antibody proteins according to the invention, wherein the aromatic amino acid in Kabat position 87 of the light chain region is a tyrosine or phenylalanine.
In a further embodiment, the present invention also pertains to antibody proteins according to the invention, wherein the amino acid in Kabat position 36 of the light chain region is selected from aromatic amino acids.
In a particular embodiment the invention relates to the specific antibody proteins that may be prepared from the individually disclosed reshaped variable regions of the light and heavy chains.
Especially light chain variable region versions A and C are particularly suitable to practice the invention because of their exceptionally high producibility, while retaining full FAP-binding specificity and achieving low immunogenicity. This holds especially true when compared to the chimeric antibody having the variable regions of F19 and the same constant regions but also when compared to light chain version B.
Therefore, in one embodiment the present invention relates to antibody proteins that contain the variable region of the light chain as set forth in SEQ ID NO:2.
In a further embodiment the invention also relates to antibody proteins, characterized in that the variable region of the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:1.
In one embodiment the present invention relates to antibody proteins that contain the variable region of the light chain as set forth in SEQ ID NO:6.
In a further embodiment the invention also relates to antibody proteins characterized in that the variable region of the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:5.
The present invention also discloses several different variable regions of the heavy chain that work particularly well with the variable regions of the light chain versions A and C in terms of improved producibility.
In one embodiment the invention relates to antibody proteins containing a variable region of the heavy chain as set forth in any one of SEQ ID NOS:8, 10, 12 and 14.
In another embodiment the invention relates to antibody proteins characterized in that the variable region of the heavy chain is encoded by a nucleotide sequence as set forth in any one of SEQ ID NOS:7, 9, 11 and 13.
In a very particular embodiment the invention relates to antibody proteins containing the variable region of the light chain as set forth in SEQ ID NO:2 and the variable region of the heavy chain as set forth in SEQ ID NO:12. Most preferably, this antibody protein additionally contains the constant region of the light chain as set forth in SEQ ID NO:20 and the constant region of the heavy chain as set forth in SEQ ID NO:22.
Thus a further aspect of the present invention is an antibody protein containing an amino acid sequence as set forth in SEQ ID NO:2. More preferably, such an antibody protein further contains an amino acid sequence as set forth in SEQ ID NO:12. More preferably, said antibody protein further contains an amino acid sequence as set forth in SEQ ID NO:20 and an amino acid sequence as set forth in SEQ ID NO:22. A further aspect of the invention is an antibody protein as described in this paragraph which is conjugated to a radioisotope, preferably 131I, 125I, 186Re, 188Re, or 90Y. An additional aspect of the present invention is a DNA molecule coding for an antibody protein as described in this paragraph. A further aspect of the invention is a host cell carrying such a DNA molecule. Accordingly, a further aspect of the invention is a method of producing an antibody protein as described in this paragraph, said method comprising the steps of cultivating such a host cell under conditions where said antibody protein is expressed by said host cell, and isolating said protein. A further aspect of the invention is a pharmaceutical composition comprising an antibody protein of the present invention and a pharmaceutically acceptable carrier.
In a further particular embodiment the invention relates to antibody proteins characterized in that the variable region of the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:1 and the variable region of the heavy chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:11.
In a further particular embodiment the invention relates to antibody proteins containing the variable region of the light chain as set forth in SEQ ID NO:2 and the variable region of the heavy chain as set forth in SEQ ID NO:8.
In a further particular embodiment the invention relates to antibody proteins characterized in that the variable region of the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:1 and the variable region of the heavy chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:7.
Humanization of the variable region of a murine antibody may be achieved employing methods known in the art. EP 0230400 discloses grafting of the CDRs of a murine variable region into the framework of a human variable region. WO 90/07861 discloses methods of reshaping a CDR-grafted variable region by introducing additional framework modifications. WO 92/11018 discloses methods of producing humanized Ig combining donor CDRs with an acceptor framework that has a high homology to the donor framework. WO 92/05274 discloses the preparation of framework mutated antibodies starting from a murine antibody. Further prior art references related to humanization of murine monoclonal antibodies are EP 0368684; EP 0438310; WO 92/07075 or WO 92/22653. Thus, the expert can produce the antibodies of the present invention starting from the publicly available murine monoclonal antibody F19 and employing techniques known in the art, e.g., from WO 92/05274; DNA molecules coding for the antibody proteins of the present invention may of course also be obtained by state-of-the-art synthetic procedures, e.g., by chemical synthesis of appropriate oligonucleotides and subsequent ligation and amplification procedures (see e.g., Frank et al., Methods Enzymol. 154:221-249 (1987)).
In a further aspect, the present invention relates to nucleic acid molecules containing the coding information for the antibody proteins according to the invention as disclosed above. Preferably, a nucleic acid molecule according to the present invention is a nucleic acid molecule containing a nucleotide sequence selected from SEQ ID NOS:1, 3, 5, 7, 9, 11, 13 or 15.
A further aspect of the present invention is a recombinant DNA vector containing the nucleotide sequence of any one of the above-mentioned nucleic acids, especially when said nucleotide sequence is operationally linked to an expression control sequence as in expression vectors. Preferred is a recombinant DNA vector, said vector being an expression vector.
A further aspect of the present invention is a host cell carrying a vector as described, especially an expression vector. Such a host cell can be a prokaryotic or eukaryotic cell. Preferably, such a host cell is a eukaryotic cell, a yeast cell, or a mammalian cell. More preferably, said host cell is a CHO (Chinese hamster ovary) cell or a COS cell.
Accordingly, a still further aspect of the present invention is a method of producing antibody proteins according to the invention. Such a method comprises the steps of:
(a) cultivating a host cell as described above under conditions where said antibody protein is expressed by said host cell, and
(b) isolating said antibody protein.
Mammalian host cells, preferably CHO or COS cells are preferred. Host cells for producing the antibody proteins of the invention may be transfected with a single vector containing the expression units for both, the light and the heavy chain (see, e.g., WO 94/11523). In one particular embodiment the method of producing antibody proteins according to the invention pertains to host cells, wherein said host cells are cotransfected with two plasmids carrying the expression units for the light and heavy chains respectively (see, e.g., EP 0481790).
The antibody proteins of the invention provide a highly specific tool for targeting therapeutic agents to the FAP antigen. Therefore, in a further aspect, the invention relates to antibody proteins according to the invention, wherein said antibody protein is conjugated to a therapeutic agent. Of the many therapeutic agents known in the art, therapeutic agents selected from the group consisting of radioisotopes, toxins, toxoids, inflammatogenic agents, enzymes, antisense molecules, peptides, cytokines, and chemotherapeutic agents are preferred. Among the radioisotopes, gamma, beta and alpha-emitting radioisotopes may be used as a therapeutic agent. xcex2-emitting radioisotopes are preferred as therapeutic radioisotopes. 186Rhenium, 188Rhenium, 131Iodine and 90Yttrium have been proven to be particularly useful xcex2-emitting isotopes to achieve localized irradiation and destruction of malignant tumor cells. Therefore, radioisotopes selected from the group consisting of 186Rhenium, 188Rhenium, 131Iodine and 90Yttrium are particularly preferred as therapeutic agents conjugated to the antibody proteins of the invention. For example, for the radioiodination of an antibody of the invention, a method as disclosed in WO 93/05804, may be employed.
A further aspect of the present invention pertains to antibody proteins according to the invention, characterized in that they are labelled. Such an FAP-specific labelled antibody allows for the localization and/or detection of the FAP antigen in vitro and/or in vivo. A label is defined as a marker that may be directly or indirectly detectable. An indirect marker is defined as a marker that cannot be detected by itself but needs a further directly detectable marker specific for the indirect marker. Preferred labels for practicing the invention are detectable markers. From the large variety of detectable markers, a detectable marker selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin is most preferred.
A further aspect of the present invention relates to antibody proteins according to the invention, characterized in that they are conjugated to an imageable agent. A large variety of imageable agents, especially radioisotopes, are available from the state of the art. For practicing the invention gamma-emitting isotopes are more preferred. Most preferred is 125Iodine.
One aspect of the present invention relates to pharmaceutical compositions containing an antibody protein according to the present invention as described above and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful for treating tumors, wherein said tumors are associated with activated stromal fibroblasts. There are two possible effector principles for an anti-tumor stroma immunotherapy that may act synergistically: (a) an unmodified (unconjugated, xe2x80x9cnakedxe2x80x9d) antibody according to the invention may induce immune destruction or inflammatory reactions in the tumor stroma while (b) an antibody conjugated to a therapeutic agent, such as for example, a radioisotope or other toxic substance, may achieve localized irradiation and destruction of the malignant tumor cells. Accordingly, a further aspect of the present invention is the use of an antibody protein as described for the manufacture of a pharmaceutical composition, especially for the treatment of tumors.
One further embodiment are pharmaceutical compositions containing an antibody protein according to the invention conjugated to a therapeutic agent as described above and a pharmaceutically acceptable carrier useful for treating tumors, wherein said tumors are associated with activated stromal fibroblasts. Another embodiment pertains to pharmaceutical compositions containing an antibody protein according to the present invention conjugated to an imageable agent as described above and a pharmaceutically acceptable carrier useful for imaging the presence of activated stromal fibroblasts in a healing wound, inflamed skin or a tumor, in a human patient. A most preferred embodiment relates to the pharmaceutical compositions mentioned above, wherein said tumors are tumors selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, invasive bladder cancers, pancreatic cancers and cancers metastatic of the brain.
In an animal or human body, it can prove advantageous to apply the pharmaceutical compositions as described above via an intravenous or other route, e.g., systemically, locally or topically to the tissue or organ of interest, depending on the type and origin of the disease or problem treated, e.g., a tumor. For example, a systemic mode of action is desired when different organs or organ systems are in need of treatment as in e.g., systemic autoimmune diseases, or allergies, or transplantations of foreign organs or tissues, or tumors that are diffuse or difficult to localize. A local mode of action would be considered when only local manifestations of neoplastic or immunologic action are expected, such as, for example local tumors.
The antibody proteins of the present invention may be applied by different routes of application known to the expert, notably intravenous injection or direct injection into target tissues. For systemic application, the intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred. A more local application can be effected subcutaneously, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily or directly in or near the tissue to be treated (connective-, bone-, muscle-, nerve-, epithelial tissue). Depending on the desired duration and effectiveness of the treatment, pharmaceutical antibody compositions may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.
For preparing suitable antibody preparations for the applications described above, the expert may use known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as, e.g., saline or corresponding plasma protein solutions are readily available. The pharmaceutical compositions may be present as lyophylisates or dry preparations, which can be reconstituted with a known injectable solution directly before use under sterile conditions, e.g., as a kit of parts. The final preparation of the antibody compositions of the present invention are prepared for injection, infusion or perfusion by mixing purified antibodies according to the invention with a sterile physiologically acceptable solution, that may be supplemented with known carrier substances or/and additives (e.g., serum albumin, dextrose, sodium bisulfite and EDTA).
The amount of the antibody applied depends on the nature of the disease. In cancer patients, the applied dose of a xe2x80x98nakedxe2x80x99 antibody may be between 0.1 and 100 mg/m2, preferably between 5 and 50 mg/m2 per application. For radiolabeled antibodies, e.g., with iodine-131, the maximally tolerated dose (MTD) has to be determined which must not be exceeded in therapeutic settings. Application of radiolabeled antibody to cancer patients may then be carried out by repeated (monthly or weekly) intravenous infusion of a dose which is below the MTD (see, e.g., Welt et al., J. Clin. Oncol. 12:1193-1203 (1994)).
Furthermore, one aspect of the present invention relates to the use of the antibody proteins according to the invention for the treatment of cancer. In a preferred embodiment the present invention relates to the use of antibody proteins according to the invention conjugated to a therapeutic agent as described above for the treatment of cancer. In another preferred embodiment the present invention relates to the use of antibody proteins according to the invention conjugated to an imageable agent for imaging activated stromal fibroblasts. In a further preferred embodiment the present invention relates to the use of labelled antibody proteins according to the invention for detecting the presence of activated stromal fibroblasts in a sample.
One aspect of the invention relates to a method of treating tumors, wherein the tumor is associated with activated stromal fibroblasts capable of specifically forming a complex with antibody proteins according to the invention, present as naked/unmodified antibodies, modified antibody proteins, such as, e.g., fusion proteins, or antibody proteins conjugated to a therapeutic agent, which comprises contacting the tumor with an effective amount of said antibodies. In a preferred embodiment the present invention relates to a method of treating tumors as mentioned above, wherein the tumor is a tumor having cancer cells selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, invasive bladder cancers, pancreatic cancers and metastatic cancers of the brain. The method of treating tumors as described above may be effected in vitro or in vivo.
A further aspect of the invention relates to a method of detecting the presence of activated stromal fibroblasts in wound healing, inflammation or in tumors, characterized in that
(a) a sample, possibly containing activated stromal fibroblasts, is contacted with an antibody protein according to the invention under conditions suitable for the formation of a complex between said antibody and antigen,
(b) detecting the presence of said complex, thereby detecting the presence of activated stromal fibroblasts in wound healing, inflammation or a tumor.
In a preferred embodiment, the present invention relates to a method of detecting the presence of activated stromal fibroblasts in a tumor, wherein the tumor is a tumor having cancer cells selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain. Most preferred antibody proteins of the invention are those which are characterized in that they are labelled as mentioned above.
A further aspect of the invention relates to a method of imaging the presence of activated stromal fibroblasts in a healing wound, inflamed tissue (rheumatoid arthritis and cirrhosis are also positive) or a tumor, in a human patient, characterized in that
(a) an antibody protein according to the present invention conjugated to an imageable agent is administered to a human patient under conditions suitable for the formation of an antibody-antigen complex,
(b) imaging any complex formed in this manner,
(c) thereby imaging the presence of activated stromal fibroblasts in a human patient.
In a preferred embodiment the present invention relates to a method of imaging the presence of activated stromal fibroblasts as described above in tumors, wherein the tumor is a tumor having cancer cells selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain.
In a further aspect the present invention relates to a method of detecting tumor-stroma, characterized in that
(a) a suitable sample is contacted with an antibody protein according to the present invention, under conditions suitable for the formation of an antibody-antigen complex,
(b) detecting the presence of any complex so formed,
(c) relating the presence of said complex to the presence of tumor-stroma.
Antibody proteins for practicing the invention are preferably labeled with a detectable marker.
In a further aspect the present invention relates to a method of imaging tumor-stroma in a human patient, which comprises
(a) administering to the patient an antibody according to the invention conjugated to an imageable agent as described above under conditions suitable for the formation of an antibody-antigen complex,
(b) imaging any complex so formed, and thereby imaging the presence of tumor-stroma in a human patient.